Fusion proteins

ABSTRACT

The invention provides a single chain, polypeptide fusion protein, comprising: a non-cytotoxic protease, or a fragment thereof, which protease or protease fragment is capable of cleaving a protein of the exocytic fusion apparatus of a target cell; a Targeting Moiety that is capable of binding to a Binding Site on the target cell, which Binding Site is capable of undergoing endocytosis to be incorporated into an endocome within the target cell; a protease cleaving site at which site the fusion protein is cleavable by the protease, wherein the protease cleavage site is located between the non-cytotoxic protease or fragment thereof and the Targeting Moiety; and the translocation domain that is capable of translocating the protease or protease fragment from within an endosome, across the endosomal membrane and into the cytosol of the target cell.

This invention relates to non-cytotoxic fusion proteins, and to thetherapeutic application thereof.

Toxins may be generally divided into two groups according to the type ofeffect that they have on a target cell. In more detail, the first groupof toxins kill their natural target cells, and are therefore known ascytotoxic toxin molecules. This group of toxins is exemplified interalia by plant toxins such as ricin, and abrin, and by bacterial toxinssuch as diphtheria toxin, and Pseudomonas exotoxin A. Cytotoxic toxinshave attracted much interest in the design of “magic bullets” (eg.immunoconjugates, which comprise a cytotoxic toxin component and anantibody that binds to a specific marker on a target cell) for thetreatment of cellular disorders and conditions such as cancer. Cytotoxictoxins typically kill their target cells by inhibiting the cellularprocess of protein synthesis.

The second group of toxins, which are known as non-cytotoxic toxins, donot (as their name confirms) kill their natural target cells.Non-cytotoxic toxins have attracted much less commercial interest thanhave their cytotoxic counterparts, and exert their effects on a targetcell by inhibiting cellular processes other than protein synthesis.Non-cytotoxic toxins are produced by a variety of plants, and by avariety of microorganisms such as Clostridium sp. and Neisseria sp.

Clostridial neurotoxins are proteins that typically have a molecularmass of the order of 150 kDa. They are produced by various species ofbacteria, especially of the genus Clostridium, most importantly C.tetani and several strains of C. botulinum, C. butyricum and C.argentinense. There are at present eight different classes of theclostridial neurotoxin, namely: tetanus toxin, and botulinum neurotoxinin its serotypes A, B, C1, D, E, F and G, and they all share similarstructures and modes of action.

Clostridial neurotoxins represent a major group of non-cytotoxic toxinmolecules, and are synthesised by the host bacterium as singlepolypeptides that are modified post-translationally by a proteolyticcleavage event to form two polypeptide chains joined together by adisulphide bond. The two chains are termed the heavy chain (H-chain),which has a molecular mass of approximately 100 kDa, and the light chain(L-chain), which has a molecular mass of approximately 50 kDa.

L-chains possess a protease function (zinc-dependent endopeptidaseactivity) and exhibit a high substrate specificity for vesicle and/orplasma membrane associated proteins involved in the exocytic process.L-chains from different clostridial species or serotypes may hydrolysedifferent but specific peptide bonds in one of three substrate proteins,namely synaptobrevin, syntaxin or SNAP-25. These substrates areimportant components of the neurosecretory machinery.

Neisseria sp., most importantly from the species N. gonorrhoeae, producefunctionally similar non-cytotoxic proteases. An example of such aprotease is IgA protease (see WO99/58571).

It has been well documented in the art that toxin molecules may bere-targeted to a cell that is not the toxin's natural target cell. Whenso re-targeted, the modified toxin is capable of binding to a desiredtarget cell and, following subsequent translocation into the cytosol, iscapable of exerting its effect on the target cell. Said re-targeting isachieved by replacing the natural Targeting Moiety (TM) of the toxinwith a different TM. In this regard, the TM is selected so that it willbind to a desired target cell, and allow subsequent passage of themodified toxin into an endosome within the target cell. The modifiedtoxin also comprises a translocation domain to enable entry of thenon-cytotoxic protease into the cell cytosol. The translocation domaincan be the natural translocation domain of the toxin or it can be adifferent translocation domain obtained from a microbial protein withtranslocation activity.

For example, WO94/21300 describes modified clostridial neurotoxinmolecules that are capable of regulating Integral Membrane Protein (IMP)density present at the cell surface of the target cell. The modifiedneurotoxin molecules are thus capable of controlling cell activity (eg.glucose uptake) of the target cell. WO96/33273 and WO99/17806 describemodified clostridial neurotoxin molecules that target peripheral sensoryafferents. The modified neurotoxin molecules are thus capable ofdemonstrating an analgesic effect. WO00/10598 describes the preparationof modified clostridial neurotoxin molecules that target mucushypersecreting cells (or neuronal cells controlling said mucushypersecreting cells), which modified neurotoxins are capable ofinhibiting hypersecretion from said cells. WO01/21213 describes modifiedclostridial neurotoxin molecules that target a wide range of differenttypes of non-neuronal target cells. The modified molecules are thuscapable of preventing secretion from the target cells. Additionalpublications in the technical field of re-targeted toxin moleculesinclude:—WO00/62814; WO00/04926; U.S. Pat. No. 5,773,586; WO93/15766;WO00/61192; and WO99/58571.

The above-mentioned TM replacement may be effected by conventionalchemical conjugation techniques, which are well known to a skilledperson. In this regard, reference is made to Hermanson, G. T. (1996),Bioconjugate techniques, Academic Press, and to Wong, S. S. (1991),Chemistry of protein conjugation and cross-linking, CRC Press.

Chemical conjugation is, however, often imprecise. For example,following conjugation, a TM may become joined to the remainder of theconjugate at more than one attachment site.

Chemical conjugation is also difficult to control. For example, a TM maybecome joined to the remainder of the modified toxin at an attachmentsite on the protease component and/or on the translocation component.This is problematic when attachment to only one of said components(preferably at a single site) is desired for therapeutic efficacy.

Thus, chemical conjugation results in a mixed population of modifiedtoxin molecules, which is undesirable.

As an alternative to chemical conjugation, TM replacement may beeffected by recombinant preparation of a single polypeptide fusionprotein (see WO98/07864). This technique is based on the in vivobacterial mechanism by which native clostridial neurotoxin (ie.holotoxin) is prepared, and results in a fusion protein having thefollowing structural arrangement:NH₂-[protease component]-[translocation component]-[TM]-COOH

According to WO98/07864, the TM is placed towards the C-terminal end ofthe fusion protein. The fusion protein is then activated by treatmentwith a protease, which cleaves at a site between the protease componentand the translocation component. A di-chain protein is thus produced,comprising the protease component as a single polypeptide chaincovalently attached (via a disulphide bridge) to another singlepolypeptide chain containing the translocation component plus TM. Whilstthe WO 98/07864 methodology follows (in terms of structural arrangementof the fusion protein) the natural expression system of clostridialholotoxin, the present inventors have found that this system may resultin the production of certain fusion proteins that have asubstantially-reduced binding ability for the intended target cell.

There is therefore a need for an alternative or improved system forconstructing a non-cytotoxic fusion protein.

The present invention addresses one or more of the above-mentionedproblems by providing a single chain, polypeptide fusion protein,comprising:—

-   -   a. a non-cytotoxic protease, or a fragment thereof, which        protease or protease fragment is capable of cleaving a protein        of the exocytic fusion apparatus in a target cell;    -   b. a Targeting Moiety that is capable of binding to a Binding        Site on the target cell, which Binding Site is capable of        undergoing endocytosis to be incorporated into an endosome        within the target cell;    -   c. a protease cleavage site at which site the fusion protein is        cleavable by a protease, wherein the protease cleavage site is        located between the non-cytotoxic protease or fragment thereof        and the Targeting Moiety; and        a translocation domain that is capable of translocating the        protease or protease fragment from within an endosome, across        the endosomal membrane and into the cytosol of the target cell.

The WO98/07864 system works well for the preparation of conjugateshaving a TM that requires a C-terminal domain for interaction with aBinding Site on a target cell. In this regard, WO98/07864 providesfusion proteins having a C-terminal domain that is “free” to interactwith a Binding Site on a target cell. The present inventors have foundthat this structural arrangement is not suitable for all TMs. In moredetail, the present inventors have found that the WO 98/07864 fusionprotein system is not optimal for TMs requiring a N-terminal domain forinteraction with a binding site on a target cell. This problem isparticularly acute with TMs that require a specific N-terminus aminoacid residue or a specific sequence of amino acid residues including theN-terminus amino acid residue for interaction with a binding site on atarget cell.

In contrast to WO98/07864, the present invention provides a system forpreparing non-cytotoxic conjugates, wherein the TM component of theconjugate has an N-terminal domain (or an intra domain sequence) that iscapable of binding to a Binding Site on a target cell.

The non-cytotoxic protease component of the present invention is anon-cytotoxic protease, or a fragment thereof, which protease orprotease fragment is capable of cleaving different but specific peptidebonds in one of three substrate proteins, namely synaptobrevin, syntaxinor SNAP-25, of the exocytic fusion apparatus. These substrates areimportant components of the neurosecretory machinery. The non-cytotoxicprotease component of the present invention is preferably a neisserialIgA protease or a fragment thereof or a clostridial neurotoxin L-chainor a fragment thereof. A particularly preferred non-cytotoxic proteasecomponent is a botulinum neurotoxin (BoNT) L-chain or a fragmentthereof.

The translocation component of the present invention enablestranslocation of the non-cytotoxic protease (or fragment thereof) intothe target cell such that functional expression of protease activityoccurs within the cytosol of the target cell. The translocationcomponent is preferably capable of forming ion-permeable pores in lipidmembranes under conditions of low pH. Preferably it has been found touse only those portions of the protein molecule capable ofpore-formation within the endosomal membrane. The translocationcomponent may be obtained from a microbial protein source, in particularfrom a bacterial or viral protein source. Hence, in one embodiment, thetranslocation component is a translocating domain of an enzyme, such asa bacterial toxin or viral protein. The translocation component of thepresent invention is preferably a clostridial neurotoxin H-chain or afragment thereof. Most preferably it is the H_(N) domain (or afunctional component thereof), wherein H_(N) means a portion or fragmentof the H-chain of a clostridial neurotoxin approximately equivalent tothe amino-terminal half of the H-chain, or the domain corresponding tothat fragment in the intact H-chain.

The TM component of the present invention is responsible for binding theconjugate of the present invention to a Binding Site on a target cell.Thus, the TM component is simply a ligand through which a conjugate ofthe present invention binds to a selected target cell.

In the context of the present invention, the target cell may be anytarget cell, though with the proviso that the target cell is not anociceptive sensory afferent such as a primary sensory afferent. Thus,the TM may bind to non-neuronal cells and/or to neuronal cells.

It is routine to confirm that a TM binds to a given target cell. Forexample, a simple radioactive displacement experiment may be employed inwhich tissue or cells representative of the target cell are exposed tolabelled (eg. tritiated) ligand in the presence of an excess ofunlabelled ligand. In such an experiment, the relative proportions ofnon-specific and specific binding may be assessed, thereby allowingconfirmation that the ligand binds to the target cell. Optionally, theassay may include one or more binding antagonists, and the assay mayfurther comprise observing a loss of ligand binding. Examples of thistype of experiment can be found in Hulme, E. C. (1990), Receptor-bindingstudies, a brief outline, pp. 303-311, In Receptor biochemistry, APractical Approach, Ed. E. C. Hulme, Oxford University Press.

The fusion proteins of the present invention generally demonstrate areduced binding affinity (in the region of up to 100-fold) for targetcells when compared with the corresponding ‘free’ TM. However, despitethis observation, the fusion proteins of the present inventionsurprisingly demonstrate good efficacy. This can be attributed to twoprincipal features. First, the non-cytotoxic protease component iscatalytic—thus, the therapeutic effect of a few such molecules israpidly amplified. Secondly, the receptors present on the target cellsneed only act as a gateway for entry of the therapeutic, and need notnecessarily be stimulated to a level required in order to achieve aligand-receptor mediated pharmacological response. Accordingly, thefusion proteins of the present invention may be administered at a dosagethat is lower that would be employed for other types of therapeuticmolecules, which are typically administered at high microgram tomilligram (even up to hundreds of milligram) quantities. In contrast,the fusion proteins of the present invention may be administered at muchlower dosages, typically at least 10-fold lower, and more typically at100-fold lower.

The TM preferably comprises a maximum of 50 amino acid residues, morepreferably a maximum of 40 amino acid residues, particularly preferablya maximum of 30 amino acid residues, and most preferably a maximum of 20amino acid residues.

Proteinase activated receptor ligands represent a preferred group of TMsof the present invention, in particular PAR1. PARs represent a uniquesubtype of 7-transmembrane receptor G-protein-coupled receptors in thatthey are proteolytically modified to expose a new extracellularN-terminus, which acts as a tethered activating ligand. PAR1 agonists(such as TFLLR) have been identified that activate their cognatereceptor.

Parathyroid hormone (PTH) also represents a preferred TM of the presentinvention. PTH is released by the parathyroid gland and binds to thePTH-1 receptor. This receptor has a widespread distribution but isparticularly abundant in PTH target tissues, predominantly the kidneyand in bone.

Thus, the most preferred TMs of the present invention are:—

LIGAND REFERENCE Protease activated C. K. Derian, B. E. Maryanoff, P.receptor Ligand Andrade-Gordon, and H-C Zhang (PAR1) DRUG DEVELOPMENTRESEARCH 59: 355 (2003) PTH Shimizu M., et al 2000, J Biol Chem. Jul 21;275(29): 21836-43

According to one embodiment of the present invention, the TM binds to amucus-secreting cell, or to a neuronal cell controlling or directingmucus secretion. More specifically, the TM bind to (a) cells thatsecrete mucins, such as epithelial goblet cells and submucosal glandmucus secreting cells, (b) cells that secrete aqueous components ofmucus, such as Clara cells and serous cells, or (c) cells that controlor direct mucus secretion, such as “sensory-efferent” C-fibres, or NANCneural system fibres. In this regard, particular mention is made to theTMs:—VIP; beta₂ adrenoreceptor agonists; gastrin-releasing peptide; andcalcitonin gene related peptide. Thus, according to this embodiment,said conjugates have therapeutic application in treating mucushypersecretion, asthma, and/or chronic obstructive pulmonary disease.

In another embodiment, the TM binds to an endocrine cell. Particularmention is made here to thyroid stimulating hormone (TSH); insulin,insulin-like growth factor; TSH releasing hormone (protirelin); FSH/LHreleasing hormone (gonadorelin); corticotrophin releasing hormone (CRH);and ACTH. Thus, according to this embodiment, said conjugates havetherapeutic application in treating:—endocrine neoplasia including MEN;thyrotoxicosis and other diseases dependent on hypersecretions from thethyroid; acromegaly, hyperprolactinaemia, Cushings disease and otherdiseases dependent on anterior pituitary hypersecretion;hyperandrogenism, chronic anovulation and other diseases associated withpolycystic ovarian syndrome.

In another embodiment the TM binds to an inflammatory cell. Particularmention here is made to ligands (i) for mast cells, such as the C4domain of the Fc IgE; (ii) for eosinophils, such as ligands to theC3a/C4a-R complement receptor, antigens reactive towards CR4 complementreceptor; (iii) for macrophages and monocytes, such as macrophagestimulating factor, (iv) for neutrophils, such as an antigen associatedwith the iC3b complement receptor, or IL8. Thus, according to thisembodiment, said conjugates have therapeutic application for treatingallergies (seasonal allergic rhinitis (hay fever), allergicconjunctivitis, vasomotor rhinitis and food allergy), eosinophilia,asthma, rheumatoid arthritis, systemic lupus erythematosus, discoidlupus erythematosus, ulcerative colitis, Crohn's disease, haemorrhoids,pruritus, glomerulonephritis, hepatitis, pancreatitis, gastritis,vasculitis, myocarditis, psoriasis, eczema, chronic radiation-inducedfibrosis, lung scarring and other fibrotic disorders.

In another embodiment, the TM binds to an exocrine cell. Particularmention here is made to pituitary adenyl cyclase activating peptide(PACAP-38). Thus, according to this embodiment, said conjugates havetherapeutic application for treating mucus hypersecretion frommucus-secreting cells located in the alimentary tract, in particularlocated in the colon.

In a further embodiment, the TM binds to an immunological cell. Mentionhere is made to the ligands:—Epstein Barr virus fragment/surfacefeature. Thus, according to this embodiment, said conjugates havetherapeutic application for treating myasthenia gravis, rheumatoidarthritis, systemic lupus erythematosus, discoid lupus erythematosus,organ transplant, tissue transplant, fluid transplant, Graves disease,thyrotoxicosis, autoimmune diabetes, haemolytic anaemia,thrombocytopenic purpura, neutropenia, chronic autoimmune hepatitis,autoimmune gastritis, pernicious anaemia, Hashimoto's thyroiditis,Addison's disease, Sjogren's syndrome, primary biliary cirrhosis,polymyositis, scleroderma, systemic sclerosis, pemphigus vulgaris,bullous pemphigoid, myocarditis, rheumatic carditis, glomerulonephritis(Goodpasture type), uveitis, orchitis, ulcerative colitis, vasculitis,atrophic gastritis, pernicious anaemia, type 1 diabetes mellitus.

In a further embodiment the TM binds to a cardiovascular cell. Mentionhere is made to thrombin and TRAP (thrombin receptor agonist peptide),and ligands that bind to cardiovascular endothelial cells such as GP1bsurface antigen-recognising antibodies. Thus, according to thisembodiment, said conjugates have therapeutic application for treatingcardiovascular conditions and/or hypertension

In a further embodiment, the TM binds to a bone cell. Mention here ismade to ligands that bind to osteoblasts for the treatment of a diseaseselected from osteopetrosis and osteoporosis include calcitonin, and toligands that bind to osteoclasts including osteoclast differentiationfactors (eg. TRANCE, or RANKL or OPGL). Thus, according to thisembodiment, said conjugates have therapeutic application for treatingbone conditions.

Linear and cyclic integrin binding sequences are a preferred group ofTMs of the present invention. Many integrins recognise the tripleArg-Gly-Asp (RGD) peptide sequence (Ruoslahti, 1996). The RGD motif isfound in over 100 proteins including fibronectin, tenascin, fibrinogenand vitronectin. The RGD-integrin interaction is exploited as aconserved mechanism of cell entry by many pathogens includingcoxsackievirus (Roivaninen et al., 1991) and adenovirus (Mathias et al.,1994). The linear and cyclic peptide sequences, PLAEIDGIEL andCPLAEIDGIELC respectively, have been shown to bind and internalise DNAin cells expressing α₉β₁ integrin (Schneider et al., 1999).

Other TMs of the present invention include those discovered by phagedisplay techniques, in particular those which target and areinternalised by human airway epithelial cells. These include, linear andcyclic THALWHT (Jost et al., 2001); LEBP-1 (QPFMQCLCLIYDASC), LEBP-2(RNVPPIFNDVYWIAF) and LEBP-3 (VFRVRPWYQSTSQS) (Wu et al., 2003);CDSAFVTVDWGRSMSLC (Florea et al., 2003); SERSMNF, YGLPHKF, PSGAARA,LPHKSMP, LQHKSMP (Writer et al., 2004); FSLSKPP, HSMQLST and STQAMFQpeptides (Rahim et al., 2003).

The protease cleavage site of the present invention allows cleavage(preferably controlled cleavage) of the fusion protein at a positionbetween the non-cytotoxic protease component and the TM component. It isthis cleavage reaction that converts the fusion protein from a singlechain polypeptide into a disulphide-linked, di-chain polypeptide.

According to a preferred embodiment of the present invention, the TMbinds via a domain or amino acid sequence that is located away from theC-terminus of the TM. For example, the relevant binding domain mayinclude an intra domain or an amino acid sequence located towards themiddle (ie. of the linear peptide sequence) of the TM. Preferably, therelevant binding domain is located towards the N-terminus of the TM,more preferably at or near to the N-terminus.

In one embodiment, the single chain polypeptide fusion may include morethan one proteolytic cleavage site. However, where two or more suchsites exist, they are different, thereby substantially preventing theoccurrence of multiple cleavage events in the presence of a singleprotease. In another embodiment, it is preferred that the single chainpolypeptide fusion has a single protease cleavage site.

The protease cleavage sequence(s) may be introduced (and/or any inherentcleavage sequence removed) at the DNA level by conventional means, suchas by site-directed mutagenesis. Screening to confirm the presence ofcleavage sequences may be performed manually or with the assistance ofcomputer software (eg. the MapDraw program by DNASTAR, Inc.).

Whilst any protease cleavage site may be employed, the following arepreferred:—

Enterokinase (DDDDK↓) Factor Xa (IEGR↓/IDGR↓) TEV(Tobacco Etch virus)(ENLYFQ↓G) Thrombin (LVPR↓GS) PreScission (LEVLFQ↓GP).

Also embraced by the term protease cleavage site is an intein, which isa self-cleaving sequence. The self-splicing reaction is controllable,for example by varying the concentration of reducing agent present.

In use, the protease cleavage site is cleaved and the N-terminal region(preferably the N-terminus) of the TM becomes exposed. The resultingpolypeptide has a TM with an N-terminal domain or an intra domain thatis substantially free from the remainder of the conjugate. Thisarrangement ensures that the N-terminal component (or intra domain) ofthe TM may interact directly with a Binding Site on a target cell.

In a preferred embodiment, the TM and the protease cleavage site aredistanced apart in the fusion protein by at most 10 amino acid residues,more preferably by at most 5 amino acid residues, and most preferably byzero amino acid residues. Thus, following cleavage of the proteasecleavage site, a conjugate is provided with a TM that has an N-terminaldomain that is substantially free from the remainder of the conjugate.This arrangement ensures that the N-terminal component of the TargetingMoiety may interact direct with a Binding Site on a target cell.

One advantage associated with the above-mentioned activation step isthat the TM only becomes susceptible to N-terminal degradation onceproteolytic cleavage of the fusion protein has occurred. In addition,the selection of a specific protease cleavage site permits selectiveactivation of the polypeptide fusion into a di-chain conformation.

Construction of the single-chain polypeptide fusion of the presentinvention places the protease cleavage site between the TM and thenon-cytotoxic protease component.

It is preferred that, in the single-chain fusion, the TM is locatedbetween the protease cleavage site and the translocation component. Thisensures that the TM is attached to the translocation domain (ie. asoccurs with native clostridial holotoxin), though in the case of thepresent invention the order of the two components is reversed vis-à-visnative holotoxin. A further advantage with this arrangement is that theTM is located in an exposed loop region of the fusion protein, which hasminimal structural effects on the conformation of the fusion protein. Inthis regard, said loop is variously referred to as the linker, theactivation loop, the inter-domain linker, or just the surface exposedloop (Schiavo et al 2000, Phys. Rev., 80, 717-766; Turton et al., 2002,Trends Biochem. Sci., 27, 552-558).

In one embodiment, in the single chain polypeptide, the non-cytotoxicprotease component and the translocation component are linked togetherby a disulphide bond. Thus, following cleavage of the protease cleavagesite, the polypeptide assumes a di-chain conformation, wherein theprotease and translocation components remain linked together by thedisulphide bond. To this end, it is preferred that the protease andtranslocation components are distanced apart from one another in thesingle chain fusion protein by a maximum of 100 amino acid residues,more preferably a maximum of 80 amino acid residues, particularlypreferably by a maximum of 60 amino acid residues, and most preferablyby a maximum of 50 amino acid residues.

In one embodiment, the non-cytotoxic protease component forms adisulphide bond with the translocation component of the fusion protein.For example, the amino acid residue of the protease component that formsthe disulphide bond is located within the last 20, preferably within thelast 10 C-terminal amino acid residues of the protease component.Similarly, the amino acid residue within the translocation componentthat forms the second part of the disulphide bond may be located withinthe first 20, preferably within the first 10 N-terminal amino acidresidues of the translocation component.

Alternatively, in the single chain polypeptide, the non-cytotoxicprotease component and the TM may be linked together by a disulphidebond. In this regard, the amino acid residue of the TM that forms thedisulphide bond is preferably located away from the N-terminus of theTM, more preferably towards to C-terminus of the TM.

In one embodiment, the non-cytotoxic protease component forms adisulphide bond with the TM component of the fusion protein. In thisregard, the amino acid residue of the protease component that forms thedisulphide bond is preferably located within the last 20, morepreferably within the last 10 C-terminal amino acid residues of theprotease component. Similarly, the amino acid residue within the TMcomponent that forms the second part of the disulphide bond ispreferably located within the last 20, more preferably within the last10 C-terminal amino acid residues of the TM.

The above disulphide bond arrangements have the advantage that theprotease and translocation components are arranged in a manner similarto that for native clostridial neurotoxin. By way of comparison,referring to the primary amino acid sequence for native clostridialneurotoxin, the respective cysteine amino acid residues are distancedapart by between 8 and 27 amino acid residues—taken from Popoff, M R &Marvaud, J-C, 1999, Structural & genomic features of clostridialneurotoxins, Chapter 9, in The Comprehensive Sourcebook of BacterialProtein Toxins. Ed. Alouf & Freer:—

‘Native’ length Serotype¹ Sequence between C-C BoNT/A1CVRGIITSKTKS----LDKGYNKALNDLC 23 BoNT/A2 CVRGIIPFKTKS----LDEGYNKALNDLC23 BoNT/B CKSVKAPG-------------------IC 8 BoNT/CCHKAIDGRS------------LYNKTLDC 15 BoNT/D CLRLTK---------------NSRDDSTC 12BoNT/E CKN-IVSVK----------GIRK---SIC 13 BoNT/FCKS-VIPRK----------GTKAPP-RLC 15 BoNT/G CKPVMYKNT----------GKSE----QC 13TeNT CKKIIPPTNIRENLYNRTASLTDLGGELC 27 ¹Information from proteolyticstrains only

The fusion protein may comprise one or more purification tags, which arelocated N-terminal to the protease component and/or C-terminal to thetranslocation component.

Whilst any purification tag may be employed, the following arepreferred:—

-   His-tag (eg. 6× histidine), preferably as a C-terminal and/or    N-terminal tag-   MBP-tag (maltose binding protein), preferably as an N-terminal tag-   GST-tag (glutathione-S-transferase), preferably as an N-terminal tag-   His-MBP-tag, preferably as an N-terminal tag-   GST-MBP-tag, preferably as an N-terminal tag-   Thioredoxin-tag, preferably as an N-terminal tag-   CBD-tag (Chitin Binding Domain), preferably as an N-terminal tag.

According to a further embodiment of the present invention, one or morepeptide spacer molecules may be included in the fusion protein. Forexample, a peptide spacer may be employed between a purification tag andthe rest of the fusion protein molecule (eg. between an N-terminalpurification tag and a protease component of the present invention;and/or between a C-terminal purification tag and a translocationcomponent of the present invention). A peptide spacer may be alsoemployed between the TM and translocation components of the presentinvention.

In accordance with a second aspect of the present invention, there isprovided a DNA sequence that encodes the above-mentioned single chainpolypeptide.

In a preferred aspect of the present invention, the DNA sequence isprepared as part of a DNA vector, wherein the vector comprises apromoter and terminator.

A variety of different spacer molecules may be employed in any of thefusion proteins of the present invention. Examples of such spacermolecules include GS15, GS20, GS25, and Hx27.

The present inventors have unexpectedly found that the fusion proteinsof the present invention may demonstrate an improved binding activityfor target cells when the size of the spacer is selected so that (inuse) the C-terminus of the TM and the N-terminus of the translocationcomponent are separated from one another by 40-105 angstroms, preferablyby 50-100 angstroms, and more preferably by 50-90 angstroms. In anotherembodiment, the preferred spacers have an amino acid sequence of 11-29amino acid residues, preferably 15-27 amino acid residues, and morepreferably 20-27 amino acid residues. Suitable spacers may be routinelyidentified and obtained according to Crasto, C. J. and Feng, J. A.(2000) May; 13(5); pp. 309-312—see alsohttp://www.fccc./edu/research/labs/feng/limker.html.

In a preferred embodiment, the vector has a promoter selected from:

Promoter Induction agent Typical induction condition tac (hybrid) IPTG0.2 mM (0.05-2.0 mM) AraBAD L-arabinose 0.2% (0.002-0.4%) T7-lacoperator IPTG 0.2 mM (0.05-2.0 mM)

The DNA construct of the present invention is preferably designed insilico, and then synthesised by conventional DNA synthesis techniques.

The above-mentioned DNA sequence information is optionally modified forcodon-biasing according to the ultimate host cell (eg. E. coli)expression system that is to be employed.

The DNA backbone is preferably screened for any inherent nucleic acidsequence, which when transcribed and translated would produce an aminoacid sequence corresponding to the protease cleave site encoded by thesecond peptide-coding sequence. This screening may be performed manuallyor with the assistance of computer software (eg. the MapDraw program byDNASTAR, Inc.).

According to a further embodiment of the present invention, there isprovided a method of preparing a non-cytotoxic agent, comprising:—

-   -   a. contacting a single-chain polypeptide fusion protein of the        invention with a protease capable of cleaving the protease        cleavage site;    -   b. cleaving the protease cleavage site, and thereby forming a        di-chain fusion protein.

This aspect provides a di-chain polypeptide, which generally mimics thestructure of clostridial holotoxin. In more detail, the resultingdi-chain polypeptide typically has a structure wherein:—

-   -   a. the first chain comprises the non-cytotoxic protease, or a        fragment thereof, which protease or protease fragment is capable        of cleaving a protein of the exocytic fusion apparatus of a        target cell;    -   b. the second chain comprises the TM and the translocation        domain that is capable of translocating the protease or protease        fragment from within an endosome, across the endosomal membrane        and into the cytosol of the target cell; and        the first and second chains are disulphide linked together.

According to a further aspect of the present invention, there isprovided use of a single chain or di-chain polypeptide of the invention,for the manufacture of a medicament for treating, preventing orameliorating a medical condition selected from the group consisting ofmucus hypersecretion, asthma, and/or chronic obstructive pulmonarydisease, endocrine neoplasia including MEN, thyrotoxicosis and otherdiseases dependent on hypersecretions from the thyroid; acromegaly,hyperprolactinaemia, Cushings disease and other diseases dependent onanterior pituitary hypersecretion; hyperandrogenism, chronic anovulationand other diseases associated with polycystic ovarian syndrome,allergies (seasonal allergic rhinitis (hay fever), allergicconjunctivitis, vasomotor rhinitis and food allergy), eosinophilia,asthma, rheumatoid arthritis, systemic lupus erythematosus, discoidlupus erythematosus, ulcerative colitis, Crohn's disease, haemorrhoids,pruritus, glomerulonephritis, hepatitis, pancreatitis, gastritis,vasculitis, myocarditis, psoriasis, eczema, chronic radiation-inducedfibrosis, lung scarring and other fibrotic disorders, mucushypersecretion from mucus-secreting cells located in the alimentarytract, in particular located in the colon, myasthenia gravis, rheumatoidarthritis, systemic lupus erythematosus, discoid lupus erythematosus,organ transplant, tissue transplant, fluid transplant, Graves disease,thyrotoxicosis, autoimmune diabetes, haemolytic anaemia,thrombocytopenic purpura, neutropenia, chronic autoimmune hepatitis,autoimmune gastritis, pernicious anaemia, Hashimoto's thyroiditis,Addison's disease, Sjogren's syndrome, primary biliary cirrhosis,polymyositis, scleroderma, systemic sclerosis, pemphigus vulgaris,bullous pemphigoid, myocarditis, rheumatic carditis, glomerulonephritis(Goodpasture type), uveitis, orchitis, ulcerative colitis, vasculitis,atrophic gastritis, pernicious anaemia, type 1 diabetes mellitus,cardiovascular conditions and/or hypertension, and bone conditions suchas osteopetrosis and osteoporosis.

According to a related aspect, there is provided a method of treating,preventing or ameliorating a medical condition or disease in a subject,comprising administering to said patient a therapeutically effectiveamount of a single chain or di-chain polypeptide of the invention,wherein the medical condition or disease is selected from the groupconsisting of mucus hypersecretion, asthma, and/or chronic obstructivepulmonary disease, endocrine neoplasia including MEN, thyrotoxicosis andother diseases dependent on hypersecretions from the thyroid;acromegaly, hyperprolactinaemia, Cushings disease and other diseasesdependent on anterior pituitary hypersecretion; hyperandrogenism,chronic anovulation and other diseases associated with polycysticovarian syndrome, allergies (seasonal allergic rhinitis (hay fever),allergic conjunctivitis, vasomotor rhinitis and food allergy),eosinophilia, asthma, rheumatoid arthritis, systemic lupuserythematosus, discoid lupus erythematosus, ulcerative colitis, Crohn'sdisease, haemorrhoids, pruritus, glomerulonephritis, hepatitis,pancreatitis, gastritis, vasculitis, myocarditis, psoriasis, eczema,chronic radiation-induced fibrosis, lung scarring and other fibroticdisorders, mucus hypersecretion from mucus-secreting cells located inthe alimentary tract, in particular located in the colon, myastheniagravis, rheumatoid arthritis, systemic lupus erythematosus, discoidlupus erythematosus, organ transplant, tissue transplant, fluidtransplant, Graves disease, thyrotoxicosis, autoimmune diabetes,haemolytic anaemia, thrombocytopenic purpura, neutropenia, chronicautoimmune hepatitis, autoimmune gastritis, pernicious anaemia,Hashimoto's thyroiditis, Addison's disease, Sjogren's syndrome, primarybiliary cirrhosis, polymyositis, scleroderma, systemic sclerosis,pemphigus vulgaris, bullous pemphigoid, myocarditis, rheumatic carditis,glomerulonephritis (Goodpasture type), uveitis, orchitis, ulcerativecolitis, vasculitis, atrophic gastritis, pernicious anaemia, type 1diabetes mellitus, cardiovascular conditions and/or hypertension, andbone conditions such as osteopetrosis and osteoporosis.

In use, the polypeptides of the present invention are typically employedin the form of a pharmaceutical composition in association with apharmaceutical carrier, diluent and/or excipient, although the exactform of the composition may be tailored to the mode of administration.Administration is preferably to a mammal, more preferably to a human.

The polypeptides may, for example, be employed in the form of an aerosolor nebulisable solution for inhalation or a sterile solution forparenteral administration, intra-articular administration orintra-cranial administration.

For treating endocrine targets, i.v. injection, direct injection intogland, or aerosolisation for lung delivery are preferred; for treatinginflammatory cell targets, i.v. injection, sub-cutaneous injection, orsurface patch administration or aerosolisation for lung delivery arepreferred; for treating exocrine targets, i.v. injection, or directinjection into or direct administration to the gland or aerosolisationfor lung delivery are preferred; for treating immunological targets,i.v. injection, or injection into specific tissues eg. thymus, bonemarrow, or lymph tissue are preferred; for treatment of cardiovasculartargets, i.v. injection is preferred; and for treatment of bone targets,i.v. injection, or direct injection is preferred. In cases of i.v.injection, this should also include the use of pump systems. In the caseof compositions for treating neuronal targets, spinal injection (eg.epidural or intrathecal) or indwelling pumps may be used.

The dosage ranges for administration of the polypeptides of the presentinvention are those to produce the desired therapeutic effect. It willbe appreciated that the dosage range required depends on the precisenature of the components, the route of administration, the nature of theformulation, the age of the patient, the nature, extent or severity ofthe patient's condition, contraindications, if any, and the judgement ofthe attending physician.

Suitable daily dosages are in the range 0.0001-1 mg/kg, preferably0.0001-0.5 mg/kg, more preferably 0.002-0.5 mg/kg, and particularlypreferably 0.004-0.5 mg/kg. The unit dosage can vary from less that 1microgram to 30 mg, but typically will be in the region of 0.01 to 1 mgper dose, which may be administered daily or preferably less frequently,such as weekly or six monthly.

A particularly preferred dosing regimen is based on 2.5 ng of fusionprotein as the 1× dose per kg patient. In this regard, preferred dosagesare in the range 1×-100× (ie. 2.5-250 ng). This dosage range issignificantly lower (ie. at least 10-fold, typically 100-fold lower)than would be employed with other types of therapeutic molecules.Moreover, the above-mentioned difference is significantly magnified whenthe same comparison is made on a molar basis—this is because the fusionproteins of the present invention have a considerably greater molecularweight than the conventional ‘small’ molecule therapeutics.

Wide variations in the required dosage, however, are to be expecteddepending on the precise nature of the components, and the differingefficiencies of various routes of administration. For example, oraladministration would be expected to require higher dosages thanadministration by intravenous injection.

Variations in these dosage levels can be adjusted using standardempirical routines for optimisation, as is well understood in the art.

Compositions suitable for injection may be in the form of solutions,suspensions or emulsions, or dry powders which are dissolved orsuspended in a suitable vehicle prior to use.

Fluid unit dosage forms are typically prepared utilising a pyrogen-freesterile vehicle. The active ingredients, depending on the vehicle andconcentration used, can be either dissolved or suspended in the vehicle.

Solutions may be used for all forms of parenteral administration, andare particularly used for intravenous injection. In preparing solutionsthe components can be dissolved in the vehicle, the solution being madeisotonic if necessary by addition of sodium chloride and sterilised byfiltration through a sterile filter using aseptic techniques beforefilling into suitable sterile vials or ampoules and sealing.Alternatively, if solution stability is adequate, the solution in itssealed containers may be sterilised by autoclaving.

Advantageously additives such as buffering, solubilising, stabilising,preservative or bactericidal, suspending or emulsifying agents and/orlocal anaesthetic agents may be dissolved in the vehicle.

Dry powders which are dissolved or suspended in a suitable vehicle priorto use may be prepared by filling pre-sterilised drug substance andother ingredients into a sterile container using aseptic technique in asterile area.

Alternatively the components (ie. agent plus inhibitor) and otheringredients may be dissolved in an aqueous vehicle, the solution issterilized by filtration and distributed into suitable containers usingaseptic technique in a sterile area. The product is then freeze driedand the containers are sealed aseptically.

Parenteral suspensions, suitable for intramuscular, subcutaneous orintradermal injection, are prepared in substantially the same manner,except that the sterile components are suspended in the sterile vehicle,instead of being dissolved and sterilisation cannot be accomplished byfiltration. The components may be isolated in a sterile state oralternatively it may be sterilised after isolation, eg. by gammairradiation.

Advantageously, a suspending agent for example polyvinylpyrrolidone isincluded in the composition/s to facilitate uniform distribution of thecomponents.

Compositions suitable for administration via the respiratory tractinclude aerosols, nebulisable solutions or microfine powders forinsufflation. In the latter case, particle size of less than 50 microns,especially less than 10 microns, is preferred. Such compositions may bemade up in a conventional manner and employed in conjunction withconventional administration devices.

Definitions Section

Targeting Moiety (TM) means any chemical structure associated with anagent that functionally interacts with a Binding Site to cause aphysical association between the agent and the surface of a target cell.In the context of the present invention, the target cell is any cellexcept a nociceptive sensory afferent. The term TM embraces any molecule(ie. a naturally occurring molecule, or a chemically/physically modifiedvariant thereof) that is capable of binding to a Binding Site on thetarget cell, which Binding Site is capable of internalisation (eg.endosome formation)—also referred to as receptor-mediated endocytosis.The TM may possess an endosomal membrane translocation function, inwhich case separate TM and Translocation Domain components need not bepresent in an agent of the present invention.

The TM of the present invention binds (preferably specifically binds) toa target cell.

The term non-cytotoxic means that the protease molecule in question doesnot kill the target cell to which it has been re-targeted.

The protease of the present invention embraces all naturally-occurringnon-cytotoxic proteases that are capable of cleaving one or moreproteins of the exocytic fusion apparatus in eukaryotic cells.

The protease of the present invention is preferably a bacterial protease(or fragment thereof). More preferably the bacterial protease isselected from the genera Clostridium or Neisseria (eg. a clostridialL-chain, or a neisserial IgA protease preferably from N. gonorrhoeae).

The present invention also embraces modified non-cytotoxic proteases,which include amino acid sequences that do not occur in nature and/orsynthetic amino acid residues, so long as the modified proteases stilldemonstrate the above-mentioned protease activity.

The protease of the present invention preferably demonstrates a serineor metalloprotease activity (eg. endopeptidase activity). The proteaseis preferably specific for a SNARE protein (eg. SNAP-25,synaptobrevin/VAMP, or syntaxin).

Particular mention is made to the protease domains of neurotoxins, forexample the protease domains of bacterial neurotoxins. Thus, the presentinvention embraces the use of neurotoxin domains, which occur in nature,as well as recombinantly prepared versions of said naturally-occurringneurotoxins.

Exemplary neurotoxins are produced by clostridia, and the termclostridial neurotoxin embraces neurotoxins produced by C. tetani(TeNT), and by C. botulinum (BoNT) serotypes A-G, as well as the closelyrelated BoNT-like neurotoxins produced by C. baratii and C. butyricum.The above-mentioned abbreviations are used throughout the presentspecification. For example, the nomenclature BoNT/A denotes the sourceof neurotoxin as BoNT (serotype A). Corresponding nomenclature appliesto other BoNT serotypes.

The term L-chain fragment means a component of the L-chain of aneurotoxin, which fragment demonstrates a metalloprotease activity andis capable of proteolytically cleaving a vesicle and/or plasma membraneassociated protein involved in cellular exocytosis.

A Translocation Domain is a molecule that enables translocation of aprotease (or fragment thereof) into a target cell such that a functionalexpression of protease activity occurs within the cytosol of the targetcell. Whether any molecule (eg. a protein or peptide) possesses therequisite translocation function of the present invention may beconfirmed by any one of a number of conventional assays.

For example, Shone C. (1987) describes an in vitro assay employingliposomes, which are challenged with a test molecule. Presence of therequisite translocation function is confirmed by release from theliposomes of K⁺ and/or labelled NAD, which may be readily monitored [seeShone C. (1987) Eur. J. Biochem; vol. 167(1): pp. 175-180].

A further example is provided by Blaustein R. (1987), which describes asimple in vitro assay employing planar phospholipid bilayer membranes.The membranes are challenged with a test molecule and the requisitetranslocation function is confirmed by an increase in conductance acrosssaid membranes [see Blaustein (1987) FEBS Letts; vol. 226, no. 1: pp.115-120].

Additional methodology to enable assessment of membrane fusion and thusidentification of Translocation Domains suitable for use in the presentinvention are provided by Methods in Enzymology Vol 220 and 221,Membrane Fusion Techniques, Parts A and B, Academic Press 1993.

The Translocation Domain is preferably capable of formation ofion-permeable pores in lipid membranes under conditions of low pH.Preferably it has been found to use only those portions of the proteinmolecule capable of pore-formation within the endosomal membrane.

The Translocation Domain may be obtained from a microbial proteinsource, in particular from a bacterial or viral protein source. Hence,in one embodiment, the Translocation Domain is a translocating domain ofan enzyme, such as a bacterial toxin or viral protein.

It is well documented that certain domains of bacterial toxin moleculesare capable of forming such pores. It is also known that certaintranslocation domains of virally expressed membrane fusion proteins arecapable of forming such pores. Such domains may be employed in thepresent invention.

The Translocation Domain may be of a clostridial origin, namely theH_(N) domain (or a functional component thereof). H_(N) means a portionor fragment of the H-chain of a clostridial neurotoxin approximatelyequivalent to the amino-terminal half of the H-chain, or the domaincorresponding to that fragment in the intact H-chain. It is preferredthat the H-chain substantially lacks the natural binding function of theH_(C) component of the H-chain. In this regard, the H_(C) function maybe removed by deletion of the H_(C) amino acid sequence (either at theDNA synthesis level, or at the post-synthesis level by nuclease orprotease treatment). Alternatively, the H_(C) function may beinactivated by chemical or biological treatment. Thus, the H-chain ispreferably incapable of binding to the Binding Site on a target cell towhich native clostridial neurotoxin (ie. holotoxin) binds.

In one embodiment, the translocation domain is a H_(N) domain (or afragment thereof) of a clostridial neurotoxin. Examples of suitableclostridial Translocation Domains include:—

Botulinum type A neurotoxin amino acid residues (449-871) Botulinum typeB neurotoxin amino acid residues (441-858) Botulinum type C neurotoxinamino acid residues (442-866) Botulinum type D neurotoxin amino acidresidues (446-862) Botulinum type E neurotoxin amino acid residues(423-845) Botulinum type F neurotoxin amino acid residues (440-864)Botulinum type G neurotoxin amino acid residues (442-863) Tetanusneurotoxin amino acid residues (458-879)

For further details on the genetic basis of toxin production inClostridium botulinum and C. tetani, we refer to Henderson et al (1997)in The Clostridia: Molecular Biology and Pathogenesis, Academic press.

The term H_(N) embraces naturally-occurring neurotoxin H_(N) portions,and modified H_(N) portions having amino acid sequences that do notoccur in nature and/or synthetic amino acid residues, so long as themodified H_(N) portions still demonstrate the above-mentionedtranslocation function.

Alternatively, the Translocation Domain may be of a non-clostridialorigin (see Table 1). Examples of non-clostridial Translocation Domainorigins include, but not be restricted to, the translocation domain ofdiphtheria toxin [O=Keefe et al., Proc. Natl. Acad. Sci. USA (1992) 89,6202-6206; Silverman et al., J. Biol. Chem. (1993) 269, 22524-22532; andLondon, E. (1992) Biochem. Biophys. Acta., 1112, pp. 25-51], thetranslocation domain of Pseudomonas exotoxin type A [Prior et al.Biochemistry (1992) 31, 3555-3559], the translocation domains of anthraxtoxin [Blanke et al. Proc. Natl. Acad. Sci. USA (1996) 93, 8437-8442], avariety of fusogenic or hydrophobic peptides of translocating function[Plank et al. J. Biol. Chem. (1994) 269, 12918-12924; and Wagner et al(1992) PNAS, 89, pp. 7934-7938], and amphiphilic peptides [Murata et al(1992) Biochem., 31, pp. 1986-1992]. The Translocation Domain may mirrorthe Translocation Domain present in a naturally-occurring protein, ormay include amino acid variations so long as the variations do notdestroy the translocating ability of the Translocation Domain.

Particular examples of viral Translocation Domains suitable for use inthe present invention include certain translocating domains of virallyexpressed membrane fusion proteins. For example, Wagner et al. (1992)and Murata et al. (1992) describe the translocation (ie. membrane fusionand vesiculation) function of a number of fusogenic and amphiphilicpeptides derived from the N-terminal region of influenza virushaemagglutinin. Other virally expressed membrane fusion proteins knownto have the desired translocating activity are a translocating domain ofa fusogenic peptide of Semliki Forest Virus (SFV), a translocatingdomain of vesicular stomatitis virus (VSV) glycoprotein G, atranslocating domain of SER virus F protein and a translocating domainof Foamy virus envelope glycoprotein. Virally encoded Aspike proteinshave particular application in the context of the present invention, forexample, the E1 protein of SFV and the G protein of the G protein ofVSV.

Use of the Translocation Domains listed in Table 1 includes use ofsequence variants thereof. A variant may comprise one or moreconservative nucleic acid substitutions and/or nucleic acid deletions orinsertions, with the proviso that the variant possesses the requisitetranslocating function. A variant may also comprise one or more aminoacid substitutions and/or amino acid deletions or insertions, so long asthe variant possesses the requisite translocating function.

TABLE 1 Translocation Amino acid domain source Residues ReferencesDiphtheria toxin 194-380 Silverman et al., 1994, J. Biol. Chem. 269,22524- 22532 London E., 1992, Biochem. Biophys. Acta., 1113, 25-51Domain II of 405-613 Prior et al., 1992, pseudomonas Biochemistry 31,3555-3559 exotoxin Kihara & Pastan, 1994, Bioconj Chem. 5, 532-538Influenza virus GLFGAIAGFIENGWEGMIDGWYG, Plank et al., 1994, J. Biol.haemagglutinin and Chem. 269, 12918-12924 Variants thereof Wagner etal., 1992, PNAS, 89, 7934-7938 Murata et al., 1992, Biochemistry 31,1986-1992 Semliki Forest Translocation domain Kielian et al., 1996, JCell virus fusogenic Biol. 134(4), protein 863-872 Vesicular 118-139 Yaoet al., 2003, Virology Stomatitis virus 310(2), 319-332 glycoprotein GSER virus F Translocation domain Seth et al., 2003, J Virol protein77(11) 6520-6527 Foamy virus Translocation domain Picard-Maureau et al.,envelope 2003, J Virol. 77(8), 4722- glycoprotein 4730

SEQ ID NOs SEQ ID1 DNA sequence of the LC/A SEQ ID2 DNA sequence of theH_(N)/A SEQ ID3 DNA sequence of the LC/B SEQ ID4 DNA sequence of theH_(N)/B SEQ ID5 DNA sequence of the LC/C SEQ ID6 DNA sequence of theH_(N)/C SEQ ID7 DNA sequence of the CP PAR1-B linker SEQ ID8 DNAsequence of the CP PTH-C linker SEQ ID9 DNA sequence of the CP PAR1-Bfusion SEQ ID10 Protein sequence of the CP PAR1-B fusion SEQ ID11 DNAsequence of the CP PTH-C fusion SEQ ID12 Protein sequence of the CPPTH-C fusion SEQ ID13 DNA sequence of the CP RGD-C linker SEQ ID14 DNAsequence of the CP RGD-C fusion SEQ ID15 Protein sequence of the CPRGD-C fusion SEQ ID16 DNA sequence of the CP cyclicRGD-C linker SEQ ID17DNA sequence of the CP cyclicRGD-C fusion SEQ ID18 Protein sequence ofthe CP cyclicRGD-C fusion SEQ ID19 DNA sequence of the CP THALWHT-Clinker SEQ ID20 DNA sequence of the CP THALWHT-C fusion SEQ ID21 Proteinsequence of the CP THALWHT-C fusion SEQ ID22 DNA sequence of the CPcyclicTHALWHT-C linker SEQ ID23 DNA sequence of the CP cyclicTHALWHT-Cfusion SEQ ID24 Protein sequence of the CP cyclicTHALWHT-C fusion SEQID25 DNA sequence of the CP ANP-C linker SEQ ID26 DNA sequence of the CPANP-C fusion SEQ ID27 Protein sequence of the CP ANP-C fusion SEQ ID28DNA sequence of the CP VIP-C linker SEQ ID29 DNA sequence of the CPVIP-C fusion SEQ ID30 Protein sequence of the CP VIP-C fusion SEQ ID31DNA sequence of the CP Gastrin releasing peptide -C linker SEQ ID32 DNAsequence of the CP Gastrin releasing peptide-C fusion SEQ ID33 Proteinsequence of the CP Gastrin releasing peptide -C fusion

EXAMPLES Example 1 Preparation of LC/B and H_(N)/B Backbone Clones

The following procedure creates the LC and H_(N) fragments for use asthe component backbone for multidomain fusion expression. This exampleis based on preparation of a serotype B based clone (SEQ ID3 and SEQID4), though the procedures and methods are equally applicable to theother serotypes (illustrated by the sequence listing for serotype A (SEQID1 and SEQ ID2) and serotype C (SEQ ID5 and SEQ ID6)).

Preparation of Cloning and Expression Vectors

pCR 4 (Invitrogen) is the chosen standard cloning vector chosen due tothe lack of restriction sequences within the vector and adjacentsequencing primer sites for easy construct confirmation. The expressionvector is based on the pMAL (NEB) expression vector, which has thedesired restriction sequences within the multiple cloning site in thecorrect orientation for construct insertion (BamHI-Sal/I-PstI-HindIII).A fragment of the expression vector has been removed to create anon-mobilisable plasmid and a variety of different fusion tags have beeninserted to increase purification options.

Preparation of Protease (Eg. LC/B) Insert

The LC/B (SEQ ID3) is created by one of two ways:

The DNA sequence is designed by back translation of the LC/B amino acidsequence (obtained from freely available database sources such asGenBank (accession number P10844) or Swissprot (accession locusBXB_CLOBO) using one of a variety of reverse translation software tools(for example EditSeq best E. coli reverse translation (DNASTAR Inc.), orBacktranslation tool v2.0 (Entelechon)). BamHI/Sal/I recognitionsequences are incorporated at the 5′ and 3′ ends respectively of thesequence maintaining the correct reading frame. The DNA sequence isscreened (using software such as MapDraw, DNASTAR Inc.) for restrictionenzyme cleavage sequences incorporated during the back translation. Anycleavage sequences that are found to be common to those required by thecloning system are removed manually from the proposed coding sequenceensuring common E. coli codon usage is maintained. E. coli codon usageis assessed by reference to software programs such as Graphical CodonUsage Analyser (Geneart), and the % GC content and codon usage ratioassessed by reference to published codon usage tables (for exampleGenBank Release 143, Sep. 13, 2004). This optimised DNA sequencecontaining the LC/B open reading frame (ORF) is then commerciallysynthesized (for example by Entelechon, Geneart or Sigma-Genosys) and isprovided in the pCR 4 vector.

The alternative method is to use PCR amplification from an existing DNAsequence with BamHI and Sal/I restriction enzyme sequences incorporatedinto the 5′ and 3′ PCR primers respectively. Complementaryoligonucleotide primers are chemically synthesised by a Supplier (forexample MWG or Sigma-Genosys) so that each pair has the ability tohybridize to the opposite strands (3′ ends pointing “towards” eachother) flanking the stretch of Clostridium target DNA, oneoligonucleotide for each of the two DNA strands. To generate a PCRproduct the pair of short oligonucleotide primers specific for theClostridium DNA sequence are mixed with the Clostridium DNA template andother reaction components and placed in a machine (the ‘PCR machine’)that can change the incubation temperature of the reaction tubeautomatically, cycling between approximately 94° C. (for denaturation),55° C. (for oligonucleotide annealing), and 72° C. (for synthesis).Other reagents required for amplification of a PCR product include a DNApolymerase (such as Taq or Pfu polymerase), each of the four nucleotidedNTP building blocks of DNA in equimolar amounts (50-200 μM) and abuffer appropriate for the enzyme optimised for Mg2+ concentration(0.5-5 mM).

The amplification product is cloned into pCR 4 using either, TOPO TAcloning for Taq PCR products or Zero Blunt TOPO cloning for Pfu PCRproducts (both kits commercially available from Invitrogen). Theresultant clone is checked by sequencing. Any additional restrictionsequences that are not compatible with the cloning system are thenremoved using site directed mutagenesis (for example using Quickchange(Stratagene Inc.)).

Preparation of Translocation (Eg. H_(N)) Insert

The H_(N)/B (SEQ ID4) is created by one of two ways:

The DNA sequence is designed by back translation of the H_(N)/B aminoacid sequence (obtained from freely available database sources such asGenBank (accession number P10844) or Swissprot (accession locusBXB_CLOBO)) using one of a variety of reverse translation software tools(for example EditSeq best E. coli reverse translation (DNASTAR Inc.), orBacktranslation tool v2.0 (Entelechon)). A PstI restriction sequenceadded to the N-terminus and XbaI-stop codon-HindIII to the C-terminusensuring the correct reading frame in maintained. The DNA sequence isscreened (using software such as MapDraw, DNASTAR Inc.) for restrictionenzyme cleavage sequences incorporated during the back translation. Anysequences that are found to be common to those required by the cloningsystem are removed manually from the proposed coding sequence ensuringcommon E. coli codon usage is maintained. E. coli codon usage isassessed by reference to software programs such as Graphical Codon UsageAnalyser (Geneart), and the % GC content and codon usage ratio assessedby reference to published codon usage tables (for example GenBankRelease 143, Sep. 13, 2004). This optimised DNA sequence is thencommercially synthesized (for example by Entelechon, Geneart orSigma-Genosys) and is provided in the pCR 4 vector.

The alternative method is to use PCR amplification from an existing DNAsequence with PstI and XbaI-stop codon-HindIII restriction enzymesequences incorporated into the 5′ and 3′ PCR primers respectively. ThePCR amplification is performed as described above. The PCR product isinserted into pCR 4 vector and checked by sequencing. Any additionalrestriction sequences that are not compatible with the cloning systemare then removed using site directed mutagenesis (for example usingQuickchange (Stratagene Inc.)).

Example 2 Preparation of a LC/B-PAR1-H_(N)/B Fusion Protein

Preparation of Linker-PAR1-Spacer Insert

The LC-H_(N) linker can be designed from first principle, using theexisting sequence information for the linker as the template. Forexample, the serotype B linker defined as the inter-domain polypeptideregion that exists between the cysteines of the disulphide bridgebetween LC and H_(N) within which proteolytic activation occurs. Thissequence information is freely available from available database sourcessuch as GenBank (accession number P10844) or Swissprot (accession locusBXB_CLOBO). It is into this linker that an Enterokinase site, PAR1 andspacer are incorporated and using one of a variety of reversetranslation software tools (for example EditSeq best E. coli reversetranslation (DNASTAR Inc.), or Backtranslation tool v2.0 (Entelechon)),the DNA sequence encoding the linker-ligand-spacer region is determined.Restriction site are then incorporated into the DNA sequence and can bearranged as BamHI-Sal/I-linker-proteasesite-PAR1-NheI-spacer-SpeI-PstI-XbaI-stop codon-HindIII (SEQ ID7). It isimportant to ensure the correct reading frame is maintained for thespacer, PAR1 and restriction sequences and that the XbaI sequence is notpreceded by the bases, TC which would result on DAM methylation. The DNAsequence is screened for restriction sequence incorporated and anyadditional sequences are removed manually from the remaining sequenceensuring common E. coli codon usage is maintained. E. coli codon usageis assessed by reference to software programs such as Graphical CodonUsage Analyser (Geneart), and the % GC content and codon usage ratioassessed by reference to published codon usage tables (for exampleGenBank Release 143, Sep. 13, 2004). This optimised DNA sequence is thencommercially synthesized (for example by Entelechon, Geneart orSigma-Genosys) and is provided in the pCR 4 vector.

Preparation of the LC/B-PAR1-H_(N)/B Fusion

In order to create the LC-linker-PAR1-spacer-H_(N) construct (SEQ ID9),the pCR 4 vector encoding the linker (SEQ ID7) is cleaved withBamHI+Sal/I restriction enzymes. This cleaved vector then serves as therecipient vector for insertion and ligation of the LC/B DNA (SEQ ID3)cleaved with BamHI+Sal/I. The resulting plasmid DNA is then cleaved withPstI+XbaI restriction enzymes and serves as the recipient vector for theinsertion and ligation of the H_(N)/B DNA (SEQ ID4) cleaved withPstI+XbaI. The final construct contains the LC-linker-PAR1-spacer-H_(N)ORF (SEQ ID9) for transfer into expression vectors for expression toresult in a fusion protein of the sequence illustrated in SEQ ID10.

Example 3 Preparation LC/C-PTH-H_(N)/C Fusion Protein

The LC-H_(N) linker can be designed using the methods described inexample two but using the C serotype linker arranged asBamHI-Sal/I-linker-protease site-PTH-NheI-spacer-SpeI-PstI-XbaI-stopcodon-HindIII (SEQ ID8). The LC/C-PTH-H_(N)/C fusion is then assembledusing the LC/C (SEQ ID5) and H_(N)/C (SEQ ID6) made using the methodsdescribed in example one and constructed using methods described inexample two. The final construct contains the LC-linker-PTH-spacer-H_(N)ORF (SEQ ID 11) for transfer into expression vectors for expression toresult in a fusion protein of the sequence illustrated in SEQ ID 12.

Example 4 Preparation and Purification of LC/C-RGD-H_(N)/C FusionProtein

The LC-H_(N) linker is designed using the methods described in Example 2but using the C serotype linker arranged as BamHI-Sal/I-linker-proteasesite-RGD-NheI-spacer-SpeI-PstI-XbaI-stop codon-HindIII (SEQ ID 13). TheLC/C-RGD-H_(N)/C fusion is then assembled using the LC/C (SEQ ID 5) andH_(N)/C (SEQ ID 6) made using the methods described in Example 1 andconstructed using methods described in Example 2. The final constructcontains the LC-linker-RGD-spacer-H_(N) ORF (SEQ ID 14) for transferinto expression vectors for expression to result in a fusion protein ofthe sequence illustrated in SEQ ID15. The resultant expression plasmid,pMAL LC/C-RGD-H_(N)/C is transformed into E. coli BL21 for recombinantprotein expression.

Expression of LC/C-RGD-H_(N)/C Fusion Protein

Expression of the LC/C-RGD-H_(N)/C fusion protein is achieved using thefollowing protocol. Inoculate 100 ml of modified TB containing 0.2%glucose and 100 μg/ml ampicillin in a 250 ml flask with a single colonyfrom the LC/C-RGD-H_(N)/C expression strain. Grow the culture at 37° C.,225 rpm for 16 hours. Inoculate 1 L of modified TB containing 0.2%glucose and 100 μg/ml ampicillin in a 2 L flask with 10 ml of overnightculture. Grow cultures at 37° C. until an approximate OD_(600nm) of 0.5is reached at which point reduce the temperature to 16° C. After 1 hourinduce the cultures with 1 mM IPTG and grow at 16° C. for a further 16hours. FIG. 1 demonstrates the expressed protein in E. coli as analysedby SDS-PAGE.

Purification of LC/C-RGD-H_(N)/C Fusion Protein

Defrost falcon tube containing 25 ml 50 mM HEPES pH 7.2 200 mM NaCl andapproximately 10 g of E. coli BL21 cell paste. Sonicate the cell pasteon ice 30 seconds on, 30 seconds off for 10 cycles at a power of 22microns ensuring the sample remains cool. Spin the lysed cells at 18 000rpm, 4° C. for 30 minutes. Load the supernatant onto a 0.1 M NiSO₄charged Chelating column (20-30 ml column is sufficient) equilibratedwith 50 mM HEPES pH 7.2 200 mM NaCl. Using a step gradient of 10 and 40mM imidazole, wash away the non-specific bound protein and elute thefusion protein with 100 mM imidazole. Dialyse the eluted fusion proteinagainst 5 L of 50 mM HEPES pH 7.2 200 mM NaCl at 4° C. overnight andmeasure the OD of the dialysed fusion protein. Add 1 unit of factor Xaper 100 μg fusion protein and incubate at 25° C. static overnight. Loadonto a 0.1 M NiSO₄ charged Chelating column (20-30 ml column issufficient) equilibrated with 50 mM HEPES pH 7.2 200 mM NaCl. Washcolumn to baseline with 50 mM HEPES pH 7.2 200 mM NaCl. Using a stepgradient of 10 and 40 mM imidazole, wash away the non-specific boundprotein and elute the fusion protein with 100 mM imidazole. Dialyse theeluted fusion protein against 5 L of 50 mM HEPES pH 7.2 200 mM NaCl at4° C. overnight and concentrate the fusion to about 2 mg/ml, aliquotsample and freeze at −20° C. Test purified protein using OD, BCA andpurity analysis. FIG. 2 demonstrates the purified protein as analysed bySDS-PAGE.

Example 5 Preparation Lc/C-cyclicRGD-H_(N)/C Fusion Protein

The LC-H_(N) linker can be designed using the methods described inExample 2 but using the C serotype linker arranged asBamHI-Sal-linker-protease site-cyclicRGD-NheI-spacer-SpeI-PstI-XbaI-stopcodon-HindIII (SEQ ID16). The LC/C-cyclicRGD-H_(N)/C fusion is thenassembled using the LC/C (SEQ ID5) and H_(N)/C (SEQ ID6) made using themethods described in Example 1 and constructed using methods describedin Example 2. The final construct contains theLC-linker-cyclicRGD-spacer-H_(N) ORF (SEQ ID17) for transfer intoexpression vectors for expression to result in a fusion protein of thesequence illustrated in SEQ ID18. The resultant expression plasmid, pMALLC/C-cyclicRGD-H_(N)/C was transformed into E. coli BL21 for recombinantprotein expression. Expression of the fusion protein was carried out asdescribed in Example 4. FIG. 1 demonstrates the protein expressed in E.coli as analysed by SDS-PAGE.

Example 6 Preparation LC/C-THALWHT-H_(N)/C Fusion Protein

The LC-H_(N) linker can be designed using the methods described inExample 2 but using the C serotype linker arranged asBamHI-Sal/I-linker-protease site-THALWHT-NheI-spacer-SpeI-PstI-XbaI-stopcodon-HindIII (SEQ ID19). The LC/C-THALWHT-H_(N)/C fusion is thenassembled using the LC/C (SEQ ID5) and H_(N)/C (SEQ ID6) made using themethods described in Example 1 and constructed using methods describedin Example 2. The final construct contains theLC-linker-THALWHT-spacer-H_(N) ORF (SEQ ID20) for transfer intoexpression vectors for expression to result in a fusion protein of thesequence illustrated in SEQ ID21. Expression of the fusion protein wascarried out as described in Example 4. FIG. 1 demonstrates the proteinexpressed in E. coli as analysed by SDS-PAGE.

The THALWHT peptide sequence given in this Example (SEQ IDs 19, 20 and21) can be exchanged with another peptide sequence found by phagedisplay techniques. For example, LEBP-1 (QPFMQCLCLIYDASC), LEBP-2(RNVPPIFNDVYWIAF) and LEBP-3 (VFRVRPWYQSTSQS) (Wu et al., 2003);CDSAFVTVDWGRSMSLC (Florea et al., 2003); SERSMNF, YGLPHKF, PSGAARA,LPHKSMP, LQHKSMP (Writer et al., 2004); FSLSKPP, HSMQLST and STQAMFQpeptides (Rahim et al., 2003).

Example 7 Preparation LC/C-cyclicTHALWHT-H_(N)/C Fusion Protein

The LC-H_(N) linker can be designed using the methods described inExample 2 but using the C serotype linker arranged asBamHI-Sal/I-linker-proteasesite-cyclicTHALWHT-NheI-spacer-SpeI-PstI-XbaI-stop codon-HindIII (SEQID22). The LC/C-cyclicTHALWHT-H_(N)/C fusion is then assembled using theLC/C (SEQ ID5) and H_(N)/C (SEQ ID6) made using the methods described inexample one and constructed using methods described in Example 2. Thefinal construct contains the LC-linker-cyclicTHALWHT-spacer-H_(N) ORF(SEQ ID23) for transfer into expression vectors for expression to resultin a fusion protein of the sequence illustrated in SEQ ID24. Expressionof the fusion protein was carried out as described in Example 4. FIG. 1demonstrates the protein expressed in E. coli as analysed by SDS-PAGE.

The THALWHT peptide sequence given in this Example (SEQ IDs 19, 20 and21) can be exchanged with another peptide sequence found by phagedisplay techniques. For example, LEBP-1 (QPFMQCLCLIYDASC), LEBP-2(RNVPPIFNDVYWIAF) and LEBP-3 (VFRVRPWYQSTSQS) (Wu et al., 2003);CDSAFVTVDWGRSMSLC (Florea et al., 2003); SERSMNF, YGLPHKF, PSGAARA,LPHKSMP, LQHKSMP (Writer et al., 2004); FSLSKPP, HSMQLST and STQAMFQpeptides (Rahim et al., 2003).

Example 8 Preparation LC/C-ANP-H_(N)/C Fusion Protein

The LC-H_(N) linker can be designed using the methods described inExample 2 but using the C serotype linker arranged asBamHI-Sal/I-linker-protease site-ANP-NheI-spacer-SpeI-PstI-XbaI-stopcodon-HindIII (SEQ ID25). The LC/C-ANP-H_(N)/C fusion is then assembledusing the LC/C (SEQ ID5) and H_(N)/C (SEQ ID6) made using the methodsdescribed in Example 1 and constructed using methods described inExample 2. The final construct contains the LC-linker-ANP-spacer-H_(N)ORF (SEQ ID26) for transfer into expression vectors for expression toresult in a fusion protein of the sequence illustrated in SEQ ID27.

Example 9 Preparation LC/C-VIP-H_(N)/C Fusion Protein

The LC-H_(N) linker can be designed using the methods described inExample 2 but using the C serotype, linker arranged asBamHI-Sal/I-linker-protease site-VIP-NheI-spacer-SpeI-PstI-XbaI-stopcodon-HindIII (SEQ ID28). The LC/C-VIP-H_(N)/C fusion is then assembledusing the LC/C (SEQ ID5) and H_(N)/C (SEQ ID6) made using the methodsdescribed in Example 1 and constructed using methods described inExample 2. The final construct contains the LC-linker-VIP-spacer-H_(N)ORF (SEQ ID29) for transfer into expression vectors for expression toresult in a fusion protein of the sequence illustrated in SEQ ID30.

The VIP sequence given in SEQ ID28 could be replaced with VIP analogueor agonist sequences. For example, [R^(15,20,21), L¹⁷]-VIP or[R^(15,20,21), L¹⁷]-VIP-GRR (Kashimoto et al., 1996; Onoue et al.,2004), [A^(2,8,9,16,19,24)]-VIP or [A^(2,8,9,16,19,24,25)]-VIP (Igarashiet al., 2005).

Example 10 Preparation LC/C-Gastrin Releasing Peptide-H_(N)/C FusionProtein

The LC-H_(N) linker can be designed using the methods described inExample 2 but using the C serotype linker arranged asBamHI-Sal/I-linker-protease site-gastrin releasingpeptide-NheI-spacer-SpeI-PstI-XbaI-stop codon-HindIII (SEQ ID34). TheLC/C-gastrin releasing peptide-H_(N)/C fusion is then assembled usingthe LC/C (SEQ ID5) and H_(N)/C (SEQ ID6) made using the methodsdescribed in Example 1 and constructed using methods described inExample 2. The final construct contains the LC-linker-gastrin releasingpeptide-spacer-H_(N) ORF (SEQ ID35) for transfer into expression vectorsfor expression to result in a fusion protein of the sequence illustratedin SEQ ID36.

Example 11 Assessment of Functionality of the LC/C-RGD-H_(N)/C FusionProtein

The functionality of the TM component of the LC/C-RGD-H_(N)/C fusionprotein (prepared according to Example 4) is assessed by a ligandbinding assay. To facilitate assessment of ligand binding, an RGDbinding peptide is synthesised in a biotinylated and non-biotinylatedform. Binding of the fusion protein is determined by a competition assaywith the biotinylated form. Briefly, NCI-H292 cells are plated into 96well plates and viable cultures established. Cells and solutions arepre-chilled to 4° C. and solutions are prepared in cell feedingmedium-plus-HEPES (50 mM). Prior to treatment, media is removed from thecells and replaced with media-plus-HEPES (500 μl per well), which isthen also removed. Labelled ligand, at ×2 the required concentration, isadded to all wells (50 μl per well). The fusion protein, at ×2 therequired concentration, is then added to wells (50 μl per well). After 1hour at 4° C., the media is removed and replaced with media+HEPES (100μl per well). This media is removed and replaced with media+HEPES (100μl per well). Cells are lysed with 100 μl per well PBS-Tween 0.1% for 5mins at 4° C. PBS-Tween is removed and cells are washed with media+HEPES(100 μl per well). This media is removed and replaced with 100 μlPBS+100 μl streptavidin-HRP per well. Cells are incubated at RTP for 20mins. The PBS+streptavidin is removed and the cells are washed withPBS-Tween. 100 μl per well of TMB is added and the cells are incubatedat 37° C. for 10 mins. 50 μl per well 2M H₂SO₄ is added and the plateread at 450 nm. Using this methodology, the ability of the TM componentof the LC/C-RGD-H_(N)/C fusion protein to bind to the cell surface isconfirmed.

DESCRIPTION OF THE FIGURES

FIG. 1—Expression of LC/C-RGD-H_(N)/C, LC/C-cyclicRGD-H_(N)/C,LC/C-THALWHT-H_(N)/C and LC/C-cyclicTHALWHT-H_(N)/C fusion proteins inE. coli.

Using the methodology outlined in Example 4, LC/C-RGD-H_(N)/C,LC/C-cyclicRGD-H_(N)/C, LC/C-THALWHT-H_(N)/C andLC/C-cyclicTHALWHT-H_(N)/C fusion proteins were expressed in E. coliBL21 cells. Briefly, 1 L of TB media containing 0.2% glucose and 100μg/ml ampicillin was inoculated with 10 ml of starter culture. Cultureswere grown at 37° C. until an approximate OD_(600nm) of 0.5 was reachedat which point the temperature was reduced to 16° C. After 1 hour thecultures were induced with 1 mM IPTG and grown for a further 16 hours.

-   -   Lane 1, LC/C-THALWHT-H_(N)/C;    -   Lane 2, LC/C-RGD-H_(N)/C;    -   Lane 3, LC/C-cyclicTHALWHT-H_(N)/C;    -   Lane 4, LC/C-cyclicRGD-H_(N)/C.

FIG. 2—Purification of a LC/C-RGD-H_(N)/C Fusion Protein

Using the methodology outlined in Example 5, a LC/C-RGD-H_(N)/C fusionprotein was purified from E. coli BL21 cells. Briefly, the solubleproducts obtained following cell disruption were applied to anickel-charged affinity capture column. Bound proteins were eluted with100 mM imidazole, treated with Factor Xa to activate the fusion proteinand remove the maltose-binding protein (MBP) tag, then re-applied to asecond nickel-charged affinity capture column. Samples from thepurification procedure were assessed by SDS-PAGE. The final purifiedmaterial in the absence and presence of reducing agent is identified inthe lanes marked [−] and [+] respectively.

REFERENCES

-   Florea et al., (2003) J. Drug Targeting 11: 383-390-   Jost et al., (2001) FEBS lett. 489: 263-269-   Lee et al., (2001) Eur. J. Biochem. 268: 2004-2012-   Mathias et al., (1994) J. Virol. 68: 6811-6814-   Rahim et al., (2003) Biotechniques 35: 317-324-   Roivaninen et al., (1991) J. Virol. 65: 4735-4740-   Ruoslahti (1996) Ann. Rev. Cell Dev. Biol. 12: 697-715-   Schneider et al., (1999) FEBS lett. 458: 329-332-   Writer et al., (2004) J. Drug Targeting 12: 185-193-   Wu et al., (2003) Gene Ther. 10: 1429-1436

The invention claimed is:
 1. A single chain, polypeptide fusion protein,comprising: a) a non-cytotoxic protease, or a fragment thereof, whichprotease or protease fragment cleaves a protein of the exocytic fusionapparatus of a target cell; b) a Targeting Moiety that binds to aBinding Site on the target cell, which Binding Site undergoesendocytosis to be incorporated into an endosome within the target cell;c) a protease cleavage site at which site the fusion protein is cleavedby a protease, wherein the protease cleavage site is located between thenon-cytotoxic protease or fragment thereof and the Targeting Moiety; andd) a translocation domain that is capable of translocating the proteaseor protease fragment from within an endosome, across the endosomalmembrane and into the cytosol of the target cell; wherein the TargetingMoiety is located between the protease cleavage site and thetranslocation domain.
 2. The fusion protein according to claim 1,wherein the Targeting Moiety and the protease cleavage site areseparated by at most 10 amino acid residues.
 3. The fusion proteinaccording to claim 1, wherein the non-cytotoxic protease is aclostridial neurotoxin L-chain.
 4. The fusion protein according to claim1, wherein the translocation domain is the H_(N) domain of a clostridialneurotoxin.
 5. The fusion protein according to claim 1, wherein theTargeting Moiety comprises at most 50 amino acid residues.
 6. The fusionprotein according to claim 1, wherein the Targeting Moiety comprises aPAR ligand or the ligand PAR1.
 7. The fusion protein according to claim1, wherein the Targeting Moiety comprises: a ligand that binds to PTH-1;a PTH peptide; a linear or cyclic integrin-binding sequence; or a tripleArg-Gly-Asp (RGD) binding sequence.
 8. The fusion protein according toclaim 1, wherein the fusion protein comprises one or more purificationtags.
 9. The fusion protein according to claim 8, wherein the one ormore purification tags are present at the N-terminal and/or C-terminalend of the fusion protein.
 10. The fusion protein according to claim 9,wherein the one or more purification tags are joined to the fusionprotein by a peptide spacer molecule.
 11. The fusion protein accordingto claim 8, wherein the one or more purification tags are joined to thefusion protein by a peptide spacer molecule.
 12. The fusion proteinaccording to claim 1, wherein the translocation domain is separated fromthe Targeting Moiety by a peptide spacer molecule.
 13. A polypeptidefusion protein comprising a polypeptide sequence selected from the groupconsisting of SEQ ID NOs: 10, 12, 15, 18, 21, 24, 27, 30, and
 33. 14. Anucleic acid encoding the polypeptide fusion protein according toclaim
 1. 15. A nucleic acid according to claim 14, wherein the nucleicacid comprises a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs: 11, 14, 17, 20, 23, 26, 29 and
 32. 16. A DNAvector, which comprises a promoter, a nucleic acid sequence according toclaim 14, and a terminator, wherein said nucleic acid sequence islocated downstream of the promoter, and said terminator is locateddownstream of the nucleic acid.
 17. A nucleic acid which iscomplementary to the nucleic acid according to claim
 14. 18. A methodfor preparing a single-chain polypeptide fusion protein, comprisingexpressing a nucleic acid sequence according to claim 14 in a host cell.19. A method of preparing a di-chain fusion protein, comprising: a.contacting a single-chain polypeptide fusion protein according to claim1 with a protease capable of cleaving the protease cleavage site; b.cleaving the protease cleavage site; and thereby forming a di-chainfusion protein.
 20. A di-chain fusion protein obtained by the method ofclaim 19, wherein the di-chain fusion protein comprises a first chainand a second chain, and wherein a. the first chain comprises thenon-cytotoxic protease, or a fragment thereof, which protease orprotease fragment cleaves a protein of the exocytic fusion apparatus ofa target cell; and, b. the second chain comprises the Targeting Moietyand the translocation domain, wherein the translocation domaintranslocates the protease or protease fragment from within an endosome,across the endosomal membrane and into the cytosol of the target cell;and the first and second chains are disulphide linked together.
 21. Acomposition comprising a fusion protein according to claim
 1. 22. Thefusion protein according to claim 1, wherein the Targeting Moiety bindsto a cell selected from the group consisting of: a mucus secreting cell;a neuronal cell controlling or directing mucus secretion; an endocrinecell; an inflammatory cell; an exocrine cell; an immunological cell; acardiovascular cell; and a bone cell.
 23. The fusion protein accordingto claim 1, wherein the Targeting Moiety comprises a ligand selectedfrom the group consisting of: TFLLR; PAR-1; PTH; VIP; a VIP analogueselected from the group consisting of [R^(15, 20, 21), L¹⁷]-VIP,[R^(15, 20, 21), L¹⁷]-VIP-GRR, [A^(2, 8, 9, 16, 19, 24)]- VIP and[A^(2, 8, 9, 16, 19, 24, 25)]- VIP; beta₂ adrenoreceptor agonists;gastrin-releasing peptide (GRP); calcitonin gene related peptide (CGRP);thyroid-stimulating, hormone (TSH); insulin; insulin-like growth factor;TSH-releasing hormone (protirelin): FSH/LH-releasing hormone(gonadorelin); corticotrophin releasing hormone (CRH);adenocorticotrophic hormone (ACTH); pituitary adenyl cyclase activatingpeptide; a ligand to the C4 domain of the Fe IgE; a ligand to theC3a/C4a -R complement receptor; an antigen reactive towards the CR4complement receptor; macrophage stimulating factor; an antigenassociated with the iC3b complement receptor; IL8; Epstein Barr virusfragment/surface feature; thrombin; thrombin receptor agonist peptide(TRAP); GP1b surface antigen-recognizing antibodies; calcitonin; theosteoclast differentiation factors TRANCE, RANKL, and OPGL; a linear orcyclic peptide selected from the group consisting of: THALWHT, LEBP-1(QPFMQCLCLIYDASC), LEBP-2 (RNVPPIFNDVYWIAF), LEBP-3 (VFRVRPWYQSTSQS),CDSAFVTVDWGRSMSLC, SERSMNF, YGLPHKF, PSGAARA, LPHKSMP, LQHKSMP, FSLSKPP,HSMQLST, and STQAMFQ; and ANP.
 24. The fusion protein according to claim1, wherein the Targeting Moiety and the protease cleavage site areseparated by at most 5 amino acid residues.
 25. The fusion proteinaccording to claim 1, wherein the Targeting Moiety comprises at most 20amino acid residues.